The phenomenon of “tendency to marry later” is generally recognized to not only reduce the reproductive period but also have the possibility of increasing disabilities associated with pregnancy/delivery, while 52.6% of all births in Japan have been taken by women in their thirties/forties (“Population Survey Report 2004” conducted by the Ministry of Health, Labour and Welfare). The upshot is that the higher child-bearing age accompanied by tendency to marry later is behind a tendency of infertility, and the estimated total number of patients under fertility treatment reported by the Ministry of Health, Labor and Welfare exceeded 460 thousand as of FY 2002 and is predicted to continue to increase also in the future. Assisted reproduction technologies, such as artificial insemination, in vitro fertilization, and microinsemination, as important breakthroughs for infertility increase year by year in the number of performed cases even though they are not covered by public health insurance, and the Research Committee of Japan Society of Obstetrics and Gynecology reports that the total number of patients undergoing in vitro fertilization and microinsemination in 2004 was 78,000 or more and this number is two times or more the total number of the same patients in 1997. The flow of tendency to marry later appears as not readily changing; thus, it is expected to hold the important key to reverse the birthrate decline to address the need of men and women desiring pregnancy at advanced ages, further increasing the degree of dependency on assisted reproduction technologies.
According to data of the Ministry of Health, Labour and Welfare summarized in 2003, men and women each have an infertility cause ratio of 50%. Defects in spermatogenesis account for 90% or more of the cause of male infertility, and the remaining 10% is ascribed to sexual dysfunction. The defects in spermatogenesis refer to a sperm count lower than the reference, a low sperm density, a poor sperm motility, a high malformation rate of sperm, and the like, and as their causes are known aging, influences of environmental hormones, lifestyle-related diseases, zinc deficiency due to an unbalanced dietary life, stress, smoking, and the like although about 60 percent of male infertility is unexplained. On the other hand, the causes of female infertility are roughly classified into 3 types: an ovulation disorder in which ovulation from the ovary does not occur, a tubal disorder in which fertilization is interrupted by the blocked oviduct after ovulation or the like, and a disorder of implantation in which implantation in the uterus cannot occur after fertilization.
A relatively simple technique adopted by mainly targeting infertility ascribed to the male side is artificial insemination and specifically involves sorting collected sperms followed by injection into the uterine cavity using a device, and thereby aiming at in vivo fertilization. A method performed in cases where it is probably difficult to carry out in vivo fertilization, such as oviduct occlusion and oligospermia/asthenospermia, is called in vitro fertilization; especially, in vitro fertilization—embryo transfer is now a most widely used method.
The administration of in vitro fertilization is roughly divided into 3 steps. First, in order to efficiently induce ovulation, various hormonal agents, ovulation inducing agents, or the like are each administered to a female patient to elicit ovulation. Next, mature ova are sucked/removed by inserting a needle into an ovarian follicle while being confirmed with a transvaginal ultrasonographic image, and fertilized by mixing the collected ova with separately collected and purified sperms in a culture solution. When the fertilization ability of the sperms is considerably weak, microfertilization is performed in which the sperms are each penetrated into an egg artificially under a microscope. In the final step, a fertilized ovum (embryo) after sorting is transferred into the uterus, thereby aiming at implantation-pregnancy.
A fertilized ovum grown to a stage of about 5 to 6 days after the start of cleavage is called a blastocyst, and an embryo grown to the blastocyst implants in the endometrium in the case of spontaneous pregnancy. Blastocyst transfer involving transferring an embryo to the uterus at a culture stage close to the timing of implantation in spontaneous pregnancy is generally considered to show an enhanced pregnancy rate compared to transfer using a cleavage-stage embryo at 2 to 3 days after fertilization.
The number of embryos transferred at a time is controversial: there is a case where a plurality of blastocysts are transferred to increase the rate of success in pregnancy, which would simultaneously elevate the possibility of multiple pregnancy, that is, take a big risk of increasing the rate of occurrence of pregnancy hypertension syndromes and complications in the mother's body as well as increasing the percentage of occurrence of abortion, premature birth, or disability. According to the view of Japan Society of Obstetrics and Gynecology announced in 2008, the transferred embryo is single in principle, and double embryo transfer is accepted for women aged 35 years or more, women whose pregnancy has failed twice or more in a row, or the like.
In the step of ovum collection, a plurality of mature unfertilized ova can be potentially collected, in which case a better blastocyst suitable for implantation needs to be selected from a plurality of embryos at a stage in which they have been grown into a state enabling transfer after fertilization, and evaluation by morphological observation is the only way presently used (FIGS. 1 and 2).
As clinical case examples showing the difficulty of selection of a blastocyst, in a case in which are obtained two good blastocysts derived from ova collected at the same cycle in the same patient and frozen at the same growth rate and at the same point in time (at 123 hours after in vitro fertilization) in an in vitro culture solution, many examples have been experienced such as in which pregnancy did not occur for the blastocyst selected for first transfer and pregnancy occurred as a result of performing second transfer using the other blastocyst, in sites of fertility treatment (FIG. 3). Despite that the two blastocysts had entirely the same evaluation results, pregnancy did not occur in the first transfer and implantation occurred in the latter transfer, showing the limitation of the current evaluation method; this is why a highly precise evaluation method correlated with improvement in the pregnancy rate has been looked forward to.
Most desirable in evaluating a blastocyst is it that noninvasive determination is possible without using an embryo itself as a test object, and a secreted material from an embryo contained in the culture solution is an important analyte meeting such requirements. Ubiquitin is identified in a report examining protein biomarkers contained in culture solutions of human and mouse embryos using a time-of-flight mass spectrometer (Non-patent Document 1); however, no relation with the pregnancy rate is described. A similar report suggests a correlation between the concentration of β-human chorionic gonadotropin (βhCG) (which is known to be produced in the syncytiotrophoblastic layer (a part of the placenta) of a fetus from immediately after conception) in an embryo culture solution and the pregnancy rate from the results of examining βhCG by an electrochemiluminescent immunoassay (ECLIA) (Non-patent Document 2); however, there is no finding that a fertilized ovum and an embryo secrete norepinephrine during development.
As an example of simply and effectively performing the sorting of a good ovum for in vitro fertilization, is publicly available a method for detecting a refractile body as an abnormal morphology in a pre-fertilized ovum (Patent Document 1), but its invasive properties into an ovum are undeniable in that it is essential for detection to expose an ovum to excitation light from a fluorescent microscope, a confocal laser microscope, or the like.
Given the economic/psychological burden on a patient, since in vitro fertilization is said to cost 250,000 to 800,000 yen or more for each fertilization, it is of extremely high significance and a deep challenge to more reliably select a blastocyst most suitable for implantation to aim for pregnancy by fewer times of transfer. Popularization of a new technology for exactly evaluating a blastocyst will relieve clinicians from the dilemma between multiple conception due to the transfer of a plurality of embryos and the enhanced pregnancy rate, and also can serve as the impetus for a patient thinking twice about fertility treatment for economic reasons to step out into the start of the treatment because it can be expected to reduce the number of in vitro fertilizations, that is, cost, required until establishment of pregnancy.